Current approaches to treating disease by administering therapeutic proteins include in vitro production of therapeutic proteins for conventional pharmaceutical delivery (e.g. intravenous, subcutaneous, or intramuscular injection, or by intranasal or intratracheal aerosol administration) and, more recently, gene therapy.
One protein which may be useful in the treatment of platelet disorders is thrombopoietin (TPO). Platelets are small (2-3 microns in diameter) anucleated cells which play an important role in primary hemostasis by adhering to and aggregating at sites of vascular damage. In addition, platelets release factors which are important components of the blood coagulation, inflammation, and wound healing pathways. Patients with very low levels of circulating platelets (thrombocytopenia) exhibit bleeding into superficial sites (e.g. skin, mucous membranes, genitourinary tract, and gastrointestinal tract) as a result of mild trauma, and are at risk for death from catastrophic hemorrhage occurring spontaneously or resulting from trauma. The physiologic role of platelets and the etiology of platelet disorders have been described (cf. Hematology: Clinical and Laboratory Practice, Eds. R. L. Bick et al., pp. 1337-1389, Mosby, St. Louis (1993); Harrison's Principles of Internal Medicine, Eds. J. D. Wilson et al., 11th Ed., pp. 1500-1505, McGraw Hill, N.Y., 1991).
Thrombocytopenia may be caused by decreased production of platelets by the bone marrow, increased sequestration of platelets in the spleen, or accelerated platelet destruction. Decreased production of platelets by the bone marrow may result from destruction of hematopoietic precursor cells by irradiation or treatment with cytotoxic agents during therapy for cancer. In addition, alcohol, estrogens, and thiazide diuretics can suppress platelet production (drug-induced thrombocytopenia). Furthermore, infiltration of the bone marrow by malignant cells and the disorders congenital amegakaryocytic hypoplasia and thrombocytopenia with absent radii (TAR syndrome) can result in decreased platelet production.
Increased splenic sequestration of platelets may occur as a result from splenomegaly associated with a variety of conditions, including liver disease, infiltration of the spleen with tumor cells as in myeloproliferative or lymphoproliferative disorders, and Gaucher's disease.
Accelerated platelet destruction and thrombocytopenia may be caused by vasculitis, hemolytic uremic syndrome, disseminated intravascular coagulation, and the presence of intravascular prosthetic devices such as cardiac valves. In addition, certain viral infections, drugs, and autoimmune disorders lead to immunologic thrombocytopenia in which platelets become coated with antibody, immune complexes, or complement and are rapidly cleared from the circulation. A number of drugs can elicit an immune response leading to immunologic thrombocytopenia, including sulfathiazole, novobiocin, para-aminosalicylate, quinidine, quinine, carbamazepine, digitoxin, arsenical drugs, and methyldopa.
Thrombocytopenia is currently treated most readily by transfusion with platelet concentrates, although corticosteroid therapy or plasmapheresis can be effective in immunologic thrombocytopenia. Treatment with platelet concentrates is severely limited by availability of suitable donors and the risk of transmission of blood-borne infectious diseases.
As an alternative to transfusion therapy, platelet deficiencies could be treated with hematopoietic growth factors which promote proliferation and maturation of megakaryocytes, the nucleated progenitor cells from which platelets are derived. Recently, cDNA clones were isolated which encode the human, mouse, and dog analogs of a protein purified from aplastic porcine plasma which displays megakaryocytopoietic activity (de Sauvage, F. J. et al. Nature 369:533-538 (1994); Lok, S. et al. Nature 369:565-568 (1994); Bartley, T. D. et al. Cell 77:1117-1124 (1994)). The encoded protein, termed thrombopoietin (TPO), stimulates proliferation and maturation of megakaryocytes and induces platelet production in vivo upon injection into experimental animals.
Methods for the production and delivery of other proteins with therapeutic properties are desirable. For example, it has been demonstrated that recombinant .beta.-interferon is an effective medication for treatment of exacerbations in patients with relapsing-remitting multiple sclerosis (MS; see Kelley, C. L. and Smeltzer, S. C. J. Neuroscience Nursing 26:52-56 (1994)). Furthermore, it has been reported that .beta.-interferon isolated from non-transfected cultured human fibroblasts may be an effective means for preventing the progression of acute non-A, non-B hepatitis to chronic disease (Omata, M. et al., Lancet 338:914-915 (1991)).
As another example, it has been demonstrated that recombinant human DNase I is an effective agent for reducing the viscosity of sputum from cystic fibrosis (CF) patients (Shak, S. et al., Proc. Natl. Acad. Sci. USA 87:9188-9192 (1990)) and for improving pulmonary function and decreasing exacerbations of respiratory disease in CF patients (Fuchs, H. J. et al., New Engl. J. Med. 331:637-642 (1994)). It has been further suggested that DNase I may be effective in improving respiratory function in patients with other respiratory diseases, such as chronic bronchitis and pneumonia (Shak, S. et al. , op. cit.).
While TPO, .beta.-interferon, and DNase I are useful, for example, in the treatment of thrombocytopenia, MS, and CF, respectively, production of therapeutic proteins using genetic engineering technology as taught in the prior art is limited to conventional recombinant DNA methods, in which the recombinant protein is purified from mammalian cells expressing an exogenous cloned gene or cDNA under the control of a suitable promoter. The exogenous DNA encoding the protein of interest is introduced into cells in the form of a viral vector, circular plasmid DNA, or linear DNA fragment. Chinese Hamster Ovary (CHO) cell lines and their derivatives (Gottesman, M. M. Meth. Enzymol. 151:3-8 (1987) or mouse cell lines, such as NSO (Galfre, G. and Milstein, C., Meth. Enzymol. 73(B): 3-46 (1981)) or P3X63Ag8.653 (Kearney, J. et al. J. Immunol. 123: 1548-1550 (1979)) are commonly used, and the production of human therapeutic proteins is thus accomplished by expression and purification of the protein from a cell of non-human origin.
In many cases, it is desirable to produce human therapeutic proteins in a human cell, for example, when it is desired that the glycosylation pattern of the protein be similar to patterns normally found on human cells. In addition, the expression of human proteins in human cells is important in the development of gene therapy methods, in which a patient's cells are engineered to produce a desired therapeutic protein to alleviate the symptoms or cure a disease.
Clearly, the development of novel methods for the production of these human proteins in human cells would be of benefit to patients, through the availability of a wider range of products with therapeutic effectiveness. One approach proposed by scientists in the field for accomplishing this goal is to use homologous recombination, or gene targeting, to introduce a cloned, exogenous regulatory element (i.e. a promoter and/or enhancer) into a cell's genome at a pre-selected site such that the regulatory element activates expression of a nearby gene, ultimately resulting in production of the protein encoded by that gene. This approach has been suggested in U.S. Pat. No. 5,272,071 and in foreign patent applications WO 91/06666, WO 91/06667 and WO 90/11354.